Electroporation cuvettes for automation

ABSTRACT

The present disclosure relates to an electroporation device that may include many electroporation units and electroporation systems that can be used in an automated environment, e.g., as one station or module in a multi-station or multi-module cell processing environment.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 16/109,156,filed Aug. 22, 2018, entitled “Electroporation Cuvettes for Automation”,now U.S. Pat. No. 10,738,327; which claims the benefit of priority toU.S. Provisional Patent Application No. 62/551,069, filed Aug. 28, 2017,and is incorporated by reference.

FIELD OF THE INVENTION

The present disclosure relates to an electroporation device that mayinclude many electroporation units and electroporation systems that canbe used in an automated environment, e.g., as one station or module in amulti-station or multi-module cell processing environment.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that thearticles and methods referenced herein do not constitute prior art underthe applicable statutory provisions.

A cell membrane constitutes the primary barrier for the transport ofmolecules and ions between the interior and the exterior of a cell.Electroporation, also known as electropermeabilization, substantiallyincreases the cell membrane permeability in the presence of a pulsedelectric field. The technique is more reproducible, universallyapplicable, and efficient than other physical, biological or chemicaltechniques for transforming and transfecting cells.

Conventional electroporation is typically conducted by exerting shortelectric pulses of defined intensity and duration to a cuvette equippedwith embedded electrodes. The electrodes are commonly fabricated out ofaluminum (Al), stainless-steel, platinum (Pt) or graphite, and arrangedin a parallel manner. A pulse generator such as special capacitordischarge equipment is required to generate the high voltage pulses. Bytuning the electric parameters, electroporation efficiency and cellviability can be optimized.

Although traditional electroporation systems have been widely used,traditional systems require a high voltage input and suffer from adverseenvironmental conditions such as electric field distortion, local pHvariation, metal ion dissolution and excess heat generation, oftenresulting in low electroporation efficiency and/or cell viability.

Accordingly, there is a need in the art for an electroporation devicethat predictably and reproducibly electroporates a variety of celltypes, can be used with off-the-shelf liquid handling devices such asair displacement pipettes, and that can be used as part of one system ina multi-system automated cell processing environment. Additionally,there is a need in the art for an electroporation device that canelectroporate many cell samples in parallel. The disclosedelectroporation devices and electroporation systems address these needs.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter. Other features, details,utilities, and advantages of the claimed subject matter will be apparentfrom the following written Detailed Description including those aspectsillustrated in the accompanying drawings and defined in the appendedclaims.

The present disclosure provides an electroporation device comprising anelectroporation cuvette with an integrated tube or “sipper” at thebottom for cell sample input and output. The electroporation cuvette ofthe electroporation unit is configured to allow for integration into anelectroporation device for automation, such that the electroporationdevice may be used as part of a larger system where transfection ortransformation of target cells is one step of a series of processes orsteps.

In specific aspects, the invention provides an automated electroporationdevice having one to many electroporation units comprising anelectroporation cuvette coupled with an adapter or engagement member atthe top that is configured for engagement with liquid handlinginstrumentation, and a “sipper” conduit at the bottom for sample intakeand output. In some specific aspects, the electroporation device isadapted to engage with an off-the-shelf pipetting instrument, e.g., anair displacement pipette. In other specific aspects, the electroporationdevices are adapted to engage with a pump, e.g., an air displacementpump or peristaltic pump. In some embodiments, the electroporationdevice may comprise a single electroporation unit configured to be usedwith, e.g., a single channel pipette as part of an automated system. Inother embodiments, the electroporation device comprises two to manyelectroporation units configured to be used with a multi-channelpipetting instrument which also may be part of an automated system. Thatis, the electroporation device may be a module used as part of a systemincluding a pipette and an electroporation station, and thiselectroporation system may be one module in a multi-module system usedfor cell processing. For example, the electroporation module may beintegrated in a multi-module system for protein production, where cellsare transformed with an expression vector, the cells are culturedfollowing transformation, and expression of a protein or proteins ofinterest are induced in the system. In another example, theelectroporation module can be integrated into an automated multi-modulesystem for cell editing, including recursive cell editing.

In one embodiment, the specification describes an electroporation modulefor electroporating cell samples comprising a pipetting device; and anelectroporation device, wherein the electroporation device comprises: ahousing that houses an engagement member and a filter; anelectroporation cuvette comprising an electroporation chamber defined bywalls and at least two electrodes wherein the electrodes are parallel toone another and wherein the electroporation chamber is in fluidcommunication with the filter; and a sipper in fluid communication withthe electroporation chamber, wherein the sipper is configured for intakeand output of the cells and/or material to be electroporated in thevessel; and wherein the engagement members of the units are configuredsuch that the electroporation device can engage with the pipettingdevice. In some aspects the electroporation module further comprises anelectroporation station; and the electroporation module furthercomprises a first reservoir disposed between the electroporation chamberand the filter, wherein the first reservoir is in fluid communicationwith the electroporation chamber; and/or in some aspects theelectroporation device may also further comprise a second reservoirdisposed between the filter and the engagement member.

In some aspects of this embodiment, the electroporation device of theelectroporation system is configured to electroporate a cell samplehaving a volume of 1 μl to 2 ml, 50 μl to 1 ml, 100 μl to 500 μl, or 200μl to 400 μl.

In some aspects, the cells in the cell sample are mammalian cells, plantcells, yeast cells, or bacteria cells, and in some aspects, the materialto be electroporated into the cells comprises nucleic acids, peptides,proteins, hormones, cytokines, chemokines, drugs, or drug precursors;most particularly, the material to be electroporated into the cells maycomprise nucleic acids.

In some aspects, the engagement member and/or the housing of theelectroporation device comprises silicone, resin, polyvinyl chloride,polyethylene, polyamide, polyethylene, polypropylene, acrylonitrilebutadiene, polycarbonate, polyetheretheketone (PEEK), polysulfone orpolyurethane, or co-polymers of these polymers. In some aspects, thewalls of the electroporation chamber comprise glass, crystal styrene orcyclic olephin co-polymers; and the electrodes of the electroporationchamber comprise aluminum, copper, titanium, aluminum, brass, silver,rhodium, gold or platinum, or graphite.

One aspect of the electroporation module provides that the pipettingdevice is an air displacement pipette.

In addition, one aspect provides that the electroporation module is anautomated electroporation cell module, and in some aspects, theautomated electroporation cell module is one module in an automatedmulti-system cell processing system.

In some aspects, the electroporation module comprises one or moremulti-channel air displacement pipettes and one or more electroporationstations; and in some aspects of the electroporation system theelectroporation device is a multi-unit electroporation device, thepipetting device is a multi-channel air displacement pipette, and theelectroporation module further comprises a multi-unit electroporationstation. Additional aspects provide that the electroporation module isautomated.

In addition, one embodiment described provides a multi-unitelectroporation device for electroporation of cell samples in parallel,the cell sample comprising cells and a material to be electroporatedinto the cells, the electroporation device comprised of at least twoelectroporation units wherein an electroporation unit comprises: ahousing that houses an engagement member and a filter; anelectroporation cuvette comprising an electroporation chamber defined bywalls and at least two electrodes, wherein the electrodes are parallelto one another and wherein the electroporation chamber is in fluidcommunication with the filter; and a sipper in fluid communication withthe electroporation chamber, wherein the sipper is configured for intakeand output of the cell sample.

In some aspects, the units of the multi-unit electroporation devicefurther comprise a first reservoir disposed between the electroporationchamber and the filter, wherein the first reservoir is in fluidcommunication with the electroporation chamber; and/or a secondreservoir disposed between the filter and the engagement member.

In some aspects one or more electroporation units are configured toelectroporate a cell sample having a volume of 1 μl to 2 ml, 50 μl to 1ml, 100 μl to 500 μl, or 200 μl to 400 μl.

In some aspects, the cells to be electroporated in the cell sample aremammalian cells, plant cells, yeast cells, or bacteria cells and in someaspects, the material to be electroporated into the cells comprisesnucleic acids, peptides, proteins, hormones, cytokines, chemokines,drugs, or drug precursors.

Often, the engagement member is configured to engage with amulti-channel pipette, and in some aspects, the multi-channel pipette isan air displacement pipette.

In some aspects, there is provided a multi-unit electroporation modulecomprising the multi-unit electroporation device, a multi-channel airdisplacement pipette, and a multi-channel electroporation station, andin some circumstances the multi-unit electroporation module isautomated. Further, the automated multi-unit electroporation module maybe one module in an automated multi-module cell processing system.

Often the electroporation units are arranged linearly, and adjacentelectroporation units share an electrode. In some aspects, theelectroporation module comprises at least 32 electroporation units, atleast 64 electroporation units or at least 96 electroporation units. Inaddition, the multi-module cell processing system comprises one or moreof the multi-unit electroporation devices.

Additionally, there is provided a multi module automated cell systemcomprising the electroporation module.

In yet another embodiment, there is provided a method forelectroporating a cell sample using the electroporation module,comprising the steps of moving the electroporation device to engage witha vessel comprising a cell sample; sipping the cell sample from thevessel through the sipper of the electroporation device into theelectroporation chamber; moving the electroporation device to theelectroporation station; engaging the electroporation device with theelectroporation station; electroporating the cell sample in theelectroporation chamber of the electroporation device; moving theelectroporation device to a position to dispense the electroporated cellsample into a vessel comprising recovery medium; and dispensing the cellsample from the electroporation chambers through the sippers and intothe vessels comprising recovery medium.

Some aspects of this embodiment further comprise the steps of ejectingthe electroporation device from the pipetting device, and in someaspects, the method further comprises repeating the moving, sipping,moving, engaging, electroporating, moving, dispensing and ejectingsteps.

These aspects and other features and advantages of the invention aredescribed below in more detail.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments taken in conjunction with the accompanyingdrawings in which:

FIG. 1 depicts an exemplary single-unit electroporation deviceconsistent with the disclosed embodiments.

FIG. 2A depicts a single-unit electroporation device engaged with anexemplary embodiment of liquid handling instrumentation. FIG. 2Billustrates a close-up of the single-unit electroporation device engagedwith the embodiment of liquid handling instrumentation shown in FIG. 2A.

FIG. 3 illustrates a close-up of the single-unit electroporation deviceengaged with the liquid handling instrumentation shown in FIG. 2B,further engaged with a vessel or tube from which the electroporationdevice “sips” the cells and material to be electroporated.

FIG. 4 depicts a single-unit electroporation device engaged with oneembodiment of liquid handling instrumentation as well as engaged withone embodiment of a single-unit electroporation station.

FIG. 5 illustrates a multi-unit electroporation device consistent withthe disclosed embodiments.

FIG. 6 is a simplified block diagram of one embodiment of a method thatmay be performed with the electroporation devices and electroporationsystems consistent with the disclosed embodiments.

FIGS. 7A-7C are simplified block diagrams of exemplary automatedmulti-module cell processing systems in which the electroporation devicemay be used.

It should be understood that the drawings are not necessarily to scale,and that like reference numbers refer to like features.

DETAILED DESCRIPTION

The practice of the techniques described herein may employ, unlessotherwise indicated, conventional techniques and descriptions molecularbiology (including recombinant techniques), cell biology, biochemistry,and genetic engineering technology, which are within the skill of thosewho practice in the art. Such conventional techniques and descriptionscan be found in standard laboratory manuals such as Green and Sambrook,Molecular Cloning: A Laboratory Manual. 4th, ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., (2014); Current Protocols inMolecular Biology, Ausubel, et al. eds., (2017); Neumann, et al.,Electroporation and Electrofusion in Cell Biology, Plenum Press, NewYork, 1989; and Chang, et al., Guide to Electroporation andElectrofusion, Academic Press, California (1992), all of which areherein incorporated in their entirety by reference for all purposes.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a cell” refers toone or more cells, and reference to “the system” includes reference toequivalent steps, methods and devices known to those skilled in the art,and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated by reference for the purpose of describing anddisclosing devices, formulations and methodologies that may be used inconnection with the presently described invention.

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in smaller ranges, and arealso encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, features and procedures well known to thoseskilled in the art have not been described in order to avoid obscuringthe invention. The terms used herein are intended to have the plain andordinary meaning as understood by those of ordinary skill in the art.

Electroporation is a widely-used method for permeabilization of cellmembranes that works by temporarily generating pores in the cellmembranes with electrical stimulation. The applications ofelectroporation include the delivery of DNA, RNA, siRNA, peptides,proteins, antibodies, drugs or other substances to a variety of cellssuch as mammalian cells (including human cells), plant cells, archea,yeasts, other eukaryotic cells, bacteria, and other cell types.Electrical stimulation may also be used for cell fusion in theproduction of hybridomas or other fused cells. During a typicalelectroporation procedure, cells are suspended in a buffer or mediumthat is favorable for cell survival. For bacterial cell electroporation,low conductance mediums, such as water, glycerol solutions and the like,are often used to reduce the heat production by transient high current.The cells and material to be electroporated into the cells (collectively“the cell sample”) is then placed in a cuvette embedded with two flatelectrodes for an electrical discharge. For example, Bio-Rad (Hercules,Calif.) makes the GENE PULSER XCELL™ line of products to electroporatecells in cuvettes. Traditionally, electroporation requires high fieldstrength.

Generally speaking, microfluidic electroporation—using cell suspensionvolumes of less than approximately 20 ml and as low as 1 μl andtypically in the 50-500 μL range—allows more precise control over atransfection or transformation process and permits flexible integrationwith other cell processing tools compared to bench-scale electroporationdevices. Microfluidic electroporation thus provides unique advantagesfor, e.g., single cell transformation, processing and analysis;multi-unit electroporation device configurations; and integratedmulti-module cell systems for processing and analysis.

The present disclosure provides electroporation devices, electroporationsystems and methods that achieve high efficiency cell electroporationwith low toxicity where the electroporation devices and systems can beintegrated with other automated cell processing tools. Theelectroporation device of the disclosure allows for multiplexing wheretwo to many electroporation units are constructed and used in parallel,and the electroporation device allows for particularly easy integrationwith robotic liquid handling instrumentation. Such automatedinstrumentation includes, but is not limited to, off-the-shelf automatedliquid handling systems from Tecan (Mannedorf, Switzerland), Hamilton(Reno, Nev.), Beckman Coulter (Fort Collins, Colo.), etc.

In specific embodiments of the electroporation devices of thedisclosure, the transformation results in greater than 30% viable cellsafter electroporation, and even greater than 35%, 40%, 45%, 50%, 55%,60%, 70%, 75%, 80%, 85%, 90%, or even 95% viable cells followingtransformation, depending on the cell type and the nucleic acids beingintroduced into the cells.

The electroporation unit(s) of the electroporation device comprises ahousing with an electroporation chamber that forms the body of thecontainer, a first electrode and a second electrode configured to engagewith an electric pulse generator in an electroporation station and wherethe electrodes define the chamber to hold the cell sample to beelectroporated, an engagement member that allows the electroporationunit to engage with a pipette or pump, a “sipper” for intake of thecells and the material to be electroporated into the cells (collectivelythe “cell sample”) and to dispense the electroporated cell sample into,e.g., recovery medium, and a filter between the engagement member andthe electroporation chamber. In certain embodiments, the electroporationdevices are autoclavable and/or disposable.

FIG. 1 depicts an exemplary single-unit electroporation deviceconsistent with the disclosed embodiments. The single-unitelectroporation device 100 comprises from top to bottom, a housing 102that encloses an engagement member 104 configured to engage with apipette such as an automatic air displacement pipette (not shown), and afilter 106. In addition to the housing 102, there is an electroporationcuvette 110 portion of the electroporation unit comprising electrodes112, and walls 114 of the electroporation chamber 116. Additionally,between the filter 106 and the electroporation chamber 116 may be placeda first optional reservoir 108, which is in fluid communication withelectroporation chamber 116 and provides an empty repository for anycell sample that may be taken in past the electroporation chamber 116.In addition, the electroporation unit may comprise another optionalreservoir 124, which is in fluid communication with the first reservoir108 (through filter 106) and is placed between filter 106 and engagementmember 104 to protect the pipette from any liquids that may make it pastthe filter 106. In fluid communication with and coupled to theelectroporation chamber 116 is sipper 118, having an end proximal 120 tothe electroporation chamber 116 and an end distal 122 from theelectroporation chamber 116. It is the distal end 122 of the sipper thatallows for uptake and dispensing of the cell sample from theelectroporation device 100.

Electroporation units of the electroporation device may be configured toelectroporate cell sample volumes of between 1 μl to 20 ml although mosttypically in the 10 μL to 1 mL range, or the 50 μL to 500 μL range. Theapproximate dimensions of the various components of the disclosedelectroporation units are given in Table 1, and dimensions, of course,will vary depending on the volume of the cell sample and thecontainer(s) that are used to contain the cells and/or material to beelectroporated.

TABLE 1 WIDTH HEIGHT VOLUME WIDTH HEIGHT VOLUME WIDTH HEIGHT VOLUME (mm)(mm) (μl) (mm) (mm) (μl) (mm) (mm) (μl) COMPONENT RANGE RANGE RANGERANGE RANGE RANGE RANGE RANGE RANGE Electroporation 0.01-100    1-5000 1-20000 0.03-50   50-2000 500-10000 0.05-30   2-500 25-4500 chamber 116First reservoir 0.1-150 0.1-250 0.5-10000  0.3-100 30-150 20-40000.5-100 0.5-100  5-2000 108 Second reservoir 0.1-250  0.2-1000 0.1-2500 0.1-150 50-400  1-1000 0.2-100 0.5-200 2-600 124 Filter 106 0.2-5000.2-500 1-3000 0.3-250 20-200 50-2500 0.5-150 0.2-80  10-2000 Sipper 1180.02-2000 0.25-2000 1-2000 0.02-1250 250-1500 1.5-1500  0.02-10  4.0-1000 2.5-1000 

Housing 102 and engagement member 104 of the electroporation units canbe made from any suitable material, including silicone, resin, polyvinylchloride, polyethylene, polyamide, polyethylene, polypropylene,acrylonitrile butadiene, polycarbonate, polyetheretheketone (PEEK),polysulfone and polyurethane, co-polymers of these and other polymers.Similarly, the walls 112 of the electroporation chamber may be made ofany suitable material including silicone, resin, glass, glass fiber,polyvinyl chloride, polyethylene, polyamide, polyethylene,polypropylene, acrylonitrile butadiene, polycarbonate,polyetheretheketone (PEEK), polysulfone and polyurethane, co-polymers ofthese and other polymers. Exemplary materials include crystal styreneand cyclic olephin co-polymers. These structures or portions thereof canbe created through various techniques, e.g., injection molding, creationof structural layers that are fused, etc.

Filter 106 can be fashioned from any suitable and preferably inexpensivematerial, including porous plastics, hydrophobic polyethylene, cotton,or glass fiberse. Sipper 118 can be made from plastics such as polyvinylchloride, polyethylene, polyamide, polyethylene, polypropylene,acrylonitrile butadiene, polycarbonate, polyetheretheketone (PEEK),polysulfone and polyurethane, co-polymers of these and other polymers,glass (such as a glass capillary), and metal tubing such as aluminum,stainless steel, or copper. Exemplary materials include crystal styreneand cyclic olephin co-polymers. The engagement member 104 is configuredto have a dimension that is compatible with the liquid handling deviceused in the electroporation system. The components of theelectroporation devices may be manufactured separately and thenassembled, or certain components of the electroporation devices may bemanufactured or molded as a single entity, with other components addedafter molding. For example, the sipper, electroporation walls, andhousing may be manufactured or molded as a single entity, with theelectrodes, filter, engagement member later added to the single entityto form the electroporation unit. Similarly, the electroporation wallsand housing may be manufactured as a single entity, with the sipper,electrodes, filter, engagement member added to the electroporation unitafter molding.

The electrodes 112 can be formed from any suitable metal, such ascopper, titanium, aluminum, brass, silver, rhodium, gold or platinum, orgraphite. An applied electric field can destroy electrodes made from ofmetals like aluminum. If a multiple use electroporation device isdesired—as opposed to a disposable, one-use device—the electrode platescan be coated with metals resistant to electrochemical corrosion.Conductive coatings like noble metals, e.g., gold, can be used toprotect the electrode plates. For example, the electroporation cuvettemay comprise a first metal electrode and a second metal electrode madefrom titanium covered with a layer of gold.

In one embodiment, the distance between the electrodes may be between0.3 mm and 50 mm. In another embodiment, the distance between theelectrodes may be between 1 mm and 20 mm, or 1 mm to 10 mm, or 2 mm to 5mm. The inner diameter of the electroporation chamber may be between 0.1mm and 10 mm. Preferably, the first metal electrode and the second metalelectrode are separated by a distance of 2-4 mm in a parallelarrangement with variations in distance less than +/−20 μm. To avoiddifferent field intensities between the electrodes, the electrodesshould by arranged in parallel with a constant distance to each otherover the whole surface of the electrodes. Furthermore, the surface ofthe electrodes should be as smooth as possible without pin holes orpeaks. Electrodes having a roughness Rz of 1 to 10 μm are preferred. Inanother embodiment of the invention, the electroporation devicecomprises at least one additional electrode which applies a groundpotential to, e.g., the sipper portion of the electroporation device.Additionally, in a multi-unit electroporation device, the electrodes mayeither be independent, standalone electrodes. Alternatively, themulti-unit electroporation device may include electrodes arranged suchthat electroporation cuvettes in adjacent electroporation units shareelectrodes, as discussed infra in relation to FIG. 5. Such multi-unitelectroporation devices may comprise, e.g., 2 or more electroporationunits, 4 or more electroporation units, 8 or more electroporation units,16 or more electroporation units, 32 or more electroporation units, 48or more electroporation units, 64 or more electroporation units, or even96 or more electroporation units preferably in an automated module andsystem.

Electroporation chambers of various shapes are convenient to manufactureand use, and an electroporation chamber for use with the disclosedelectroporation device may use any suitable shape. For example, arectangular container, a square container, a cylindrical container, aconical container, or container with other shapes are suitable.

FIG. 2A depicts a single-unit electroporation device engaged with oneembodiment of liquid handling instrumentation. The single-unitelectroporation device comprises from top to bottom, a housing 102 thatencloses an engagement member 104 configured to engage with the shaft132 of a pipette 130 such as an automatic air displacement pipette.Coupled to the housing 102, there is an electroporation cuvette 110portion of the electroporation device 100. The electroporation chamber(not labeled, but contained within the interior of the electroporationcuvette 110) is in fluid communication with sipper 118.

FIG. 2B illustrates a close-up of the single-unit electroporation deviceengaged with the embodiment of liquid handling instrumentation shown inFIG. 2A. In FIG. 2B, the pipette is not shown; however, the shaft 132 ofthe pipette is shown engaged with the engagement member 104 withinhousing 102. Also shown is filter 106, electroporation cuvette 110(which as shown in FIG. 1 as comprising at least two electrodes, andwalls defining an electroporation chamber, none of which are labeled inFIG. 2B). Sipper 118 is coupled to and in fluid communication with theelectroporation chamber within the electroporation cuvette 110.

The pipettes that may be employed with the disclosed electroporationdevice include air displacement pipettes, which use air displacement toaspirate and dispense fluids, and in particular automated airdisplacement pipettes used in robotic systems. For example, theelectroporation device of the disclosure can be integrated with Tecan'sCavro air displacement pipettor (ADP), or the Hamilton Z-ExcursionUniversal Sampler (ZEUS), a fully automated, self-contained pipettingmodule. In addition, the Beckman Coulter Biomek i5 and i7 AutomatedWorkstations utilize piston-driven air displacement pipettes to handlevolumes of liquid in the microliter scale pipettes. Air displacementpipettes offer accurate pipetting performance from 1 to 1,000 When usedwith the disclosed electroporation device, the pipette will oftencomprise an ejector system that can automatically eject a usedelectroporation device after the electroporated cells have beendeposited into the recovery medium.

The electroporation devices of the present disclosure can be configuredto be multi-unit electroporation devices that comprise two to manyelectroporation units in parallel so that they can be used, e.g., withoff-the-shelf multi-channel pipettes; for example, a multi-unitelectroporation device may be configured with 4 or 6 electroporationunits in parallel to be used with a 4- or 6-channel pipette device forinput or output of cells and/or reagents, e.g., for use with a 24-wellculture plate; a multi-unit electroporation device may be configuredwith 8 electroporation units in parallel in parallel to be used with an8-channel pipette device for input and/or output of cells or reagents,e.g., for use with a 48- or 96-well culture plate; a multi-unitelectroporation device may be configured with 12 electroporation unitsin parallel to be used with a 12-channel pipette device for input oroutput of cells and/or reagents, e.g., for use with a 128-well cultureplate; or any other desired configuration.

In lieu of a pipette arrangement where a pipette is used to both intakeand evacuate a cell sample into the electroporation cuvette (includingthe disclosed embodiment of an automated air displacement pipette), aperistaltic pump or a vacuum pump may be employed. Accordingly, theelectroporation chamber is in communication with a pump that intakes thecell sample into the electroporation chamber and evacuates the cellsafter electroporation into a tube containing recovery medium.

FIG. 3 illustrates a close-up of the single-unit electroporation deviceengaged with the liquid handling instrumentation shown in FIG. 2B,further engaged with a tube from which the electroporation device “sips”the cells and material to be electroporated. In FIG. 3 like in FIG. 2B,the pipette is not shown; however, the shaft 132 of a pipette is shown,engaged with the engagement member 104 within housing 102 of thesingle-unit electroporation device. Also shown is filter 106,electroporation cuvette 110 (which as shown in FIG. 1 as comprising atleast two electrodes, and walls defining an electroporation chamber,none of which are labeled in FIG. 3). Sipper 118 is coupled to and influid communication with the electroporation chamber within theelectroporation cuvette 110 at its proximal end 120, and is engaged withtube 140, where the distal end 122 of sipper 118 is fully immersed intube 140. In one exemplary embodiment, cells and a material to beelectroporated into the cells (collectively, a “cell sample”) arecontained in tube 140. Alternatively, the cells and the material to beelectroporated into the cells are contained in different tubes and thesipper 118 engages with two (or more) different tubes to fill theelectroporation chamber of the electroporation cuvette with a cellsample.

The cells that may be electroporated with the disclosed electroporationdevices include mammalian cells (including human cells), plant cells,yeasts, other eukaryotic cells, bacteria, archaea, and other cell types.The volume of the cell sample that can be electroporated with thedisclosed electroporation devices is from 1 μl to 2 ml, or 50 μl to 1ml, or 100 μl to 500 μl, or 200 μl to 400 μl.

The medium or buffer used to suspend the cells and material to beelectroporated into the cells for the electroporation process may be anysuitable medium or buffer, such as MEM, DMEM, IMDM, RPMI, Hanks', PBSand Ringer's solution. For electroporation of most eukaryotic cells, themedium or buffer usually contains salts to maintain a proper osmoticpressure. The salts in the medium or buffer also render the mediumconductive. For electroporation of very small prokaryotic cells such asbacteria, sometimes water is used as a low conductance medium to allow avery high electric field strength. In that case, the charged moleculesto be delivered still render water based medium more conductive than thelipid-based cell membranes and the medium may still be roughlyconsidered as conductive especially compared to cell membranes.

The compound to be electroporated into the cells of choice can be anycompound known in the art to be useful for electroporation, such asnucleic acids, oligonucleotides, polynucleotides, DNA, RNA, peptides,proteins and small molecules like hormones, cytokines, chemokines,drugs, or drug precursors.

FIG. 4 depicts an exemplary single-unit electroporation device engagedwith one embodiment of liquid handling instrumentation (e.g., anautomated air displacement pipette) as well as engaged with oneembodiment of a single-unit electroporation station where collectivelythese components—electroporation device, liquid handlinginstrumentation, and electroporation station—are an “electroporationmodule” 160. Also seen is electroporation cuvette 110 of the single-unitelectroporation device that has been inserted into a hole or openingwithin electroporation station 150. The hole in electroporation station150 is configured to precisely engage with the electroporation cuvette110, such that the electrodes (not labeled) of electroporation cuvette110 engage with the electrical contacts 152 of the electroporationstation 150. Also seen is sipper 118.

The electroporation station 150 generates the electrical pulse toelectroporate the cells via contacts 152, where the contacts are inelectrical communication with the electrodes of the electroporationcuvette 110. The electroporation station may generate one or severaldifferent pulse forms such as a sequence of high energy pulses to openthe pores of the cell membrane, and a series of lower energy pulses totransport the material to be electroporated into the cells. There aremany different pulse forms that may be employed with the electroporationcuvette, including exponential decay waves, square or rectangular waves,arbitrary wave forms, or a selected combination of wave forms. The pulseforms for electroporation may be predetermined based on cell type, thesize and configuration of the electroporation chamber and of theelectrodes therein, and/or other parameters. The electroporation stationthus preferably comprises on-board electronics to deliver thepredetermined pulses for electroporation. One type of common pulse formis the exponential decay wave, typically made by discharging a loadedcapacitor to the cell sample. The exponential decay wave can be madeless steep by linking an inductor to the cell sample so that the initialpeak current can be attenuated. When multiple waveforms in a specifiedsequence are used, they can be in the same direction (direct current) ordifferent directions (alternating current). Using alternating currentcan be beneficial in that two topical surfaces of a cell instead of justone can be used for molecular transport, and alternating current canprevent electrolysis. The pulse generator can be controlled by a digitalor analog panel.

It is important to use voltage sufficient for achieving electroporationof material into the cells, but not too much voltage as too much powerwill decrease cell viability. For example, to electroporate a suspensionof a human cell line, 200 volts is needed for a 0.2 ml sample in a 4mm-gap cuvette with exponential discharge from a capacitor of about 1000g. However, if the same 0.2 ml cell suspension is placed in a longercontainer with 2 cm electrode distance (5 times of cuvette gapdistance), the voltage required would be 1000 volts, but a capacitor ofonly 40 μF (1/25 of 1000 μF) is needed because the electric energy froma capacitor follows the equation of:E=0.5U ² Cwhere E is electric energy, U is voltage and C is capacitance.Therefore, a high voltage pulse generator is easy to manufacture becauseit needs a much smaller capacitor to store a similar amount of energy.Similarly, it would not be difficult to generate other wave forms ofhigher voltages.

FIG. 5 illustrates a multi-unit electroporation device 200 consistentwith the disclosed embodiments. In this embodiment, electroporationunits 202, 204, 206, 208, 210, 212, 214, 216, 218, 220 of theelectroporation device 200 are arranged linearly, in parallel. Theelectroporation unit comprises an electroporation cuvette 110, coupledwith a housing 112 comprising an adapter or engagement member 104 at thetop that is configured for engagement with liquid handlinginstrumentation, and a “sipper” 118 conduit at the bottom for sampleintake and output. In this embodiment where the electroporation unitsare arranged linearly, adjacent electroporation cuvettes shareelectrodes, for example, such that the negative electrode ofelectroporation units 202 and 204 is shared, the positive electrode ofelectroporation units 204 and 206 is shared, that the negative electrodeof electroporation units 206 and 208 is shared, and so on. Themulti-unit electroporation device is configured—and the engagementmembers 104 of the electroporation unit 202, 204, etc. are configured—toengage with, e.g., the shaft of a multi-channel pipette (not shown).

FIG. 6 is a simplified block diagram of an exemplary embodiment of amethod 600 that can be performed with the electroporation device andelectroporation system consistent with the disclosed embodiments. Atstep 602, a liquid handling device (e.g., pipette) engages with anelectroporation device, such as that depicted in FIG. 1 or FIG. 5. In arobotic environment, one to many electroporation devices such as thesingle-unit electroporation device depicted in FIG. 1 may be available(e.g., lined up) for engagement with one or more liquid handlingdevices, alternatively, a multi-unit electroporation device, such asthat depicted in FIG. 5, engages with a multi-channel pipette. At step604, the combination liquid handling device and electroporation devicemoves to engage with a tube(s) containing, e.g., cells and material tobe electroporated into the cells. Alternatively, the cells and thematerial to be electroporated into the cells may be in separate tubes,and the combination liquid handling device and electroporation devicewill take in the cells and material to be electroporated into the cellsfrom separate tubes. The sipper component of the electroporation devicethen “sips” or takes up the liquid from the tube up into theelectroporation chamber of the electroporation cuvette at step 606. Asseen in FIGS. 1, 3, and 5, the electroporation chamber is encompassed bythe walls of the electroporation chamber, and two of the walls of theelectroporation chamber are adjacent to the two electrodes which areparallel to one another.

At step 608, the combination liquid handling device and electroporationdevice moves to engage with the electroporation station, which suppliesthe power to electroporate the cell sample(s) that is within theelectroporation chamber of the electroporation device. The combinationof the liquid handling device, electroporation device, andelectroporation station is referred to herein as an electroporationmodule. After the electroporation cuvette of the electroporation deviceis firmly seated within the electroporation station such that theelectrical contacts of the electroporation station are electricallyengaged with the electrodes of the electroporation device, theelectroporation station provides one or a series of electrical pulses tothe cell sample at step 610. As discussed supra, the electrical pulsesmay encompass one or several different pulse forms. In many embodiments,the electroporation station comprises on-board electronics which cancontrol the type, strength and duration of the pulse forms delivered tothe cell sample in a predetermined manner. The type of electronics boardused in the electroporation station may include but is not limited to,e.g., a capacity discharge board, or a voltage amplifying board. At step612, the combination liquid handling device and electroporation devicemoves to a dispensing station where the electroporated cells are clearedfrom the device, often into recovery medium to allow the pores of thecells to close and the cell membrane to recover from the electroporationprocess (step 614).

Once recovered, the cells then may be used in further processingprocedures such as enrichment and/or depletion of certain subsets ofcells, and/or cell culturing. The further processing procedures may beperformed as another part of an automated system that comprises two tomany automated processes. Alternatively, a second material to beelectroporated into the cells may be “sipped” and another round ofelectroporation pulses may be delivered to the cells with or without arecovery, washing, culturing or separation step. Once the electroporatedcell sample(s) has been dispensed, the pipette ejects theelectroporation device at step 616. As stated previously, in certainembodiments the electroporation device is disposable, and thus theelectroporation device may be discarded. In other embodiments theelectroporation devices are reusable and can be autoclaved between uses.

To electroporate more cells (i.e., different cells, more cells of thesame type but with different materials to be electroporated, oradditional volumes of the same cell sample), the process of filling,electroporation and clearance can be repeated (as shown by referencenumber 618).

The electroporation devices and electroporation modules of the inventionmay be a component of a broader automated cell multi-module system thatcan be utilized for various purposes involving the transformation andcapture of live cells. For example, the electroporation device may beintegrated into an automated multi-module system for protein production,where the cells are transformed with an expression vector, the cells arecultured following transformation, and the expression of a protein orproteins of interest are induced in the system. In another example, theelectroporation device may be integrated into an electroporation modulewhich is a part of an automated multi-module system for cell engineeringand selection to identify the transformed cells, with the selectionprocess occurring on the automated system. In specific aspects, theelectroporation device and module can be integrated into an automatedsystem for cell editing, including recursive cell editing. Suchautomated multi-module systems include but are not limited to thosedescribed, e.g., in U.S. Ser. No. 16/024,816, filed Jun. 30, 2018; U.S.Ser. No. 16/024,831, filed Jun. 30, 2018, U.S. Ser. No. 62/657,651,filed Apr. 13, 2018; and U.S. Ser. No. 62/657,654, filed May 14, 2018,all of which are incorporated by reference herein for all purposes.

The system may also include pressure sources and/or regulators thatcontrol the flow of liquids in the system and control the rate andvolume of electroporation.

The electroporation devices of the disclosure can allow for a high rateof cell transformation in a relatively short amount of time. The rate ofcell transformation is dependent on the cell type and the number ofcells being transformed. For example, for E. Coli, the electroporationdevices can provide a cell transformation rate of 1 to 10¹⁰ cells persecond, 10⁴ to 10⁷ per second, 10⁵ to 10⁸ per second, or 10⁶ to 10⁹ persecond. The electroporation devices also allow transformation of batchesof cells ranging from 1 cell to 10¹⁰ cells in a single transformationprocedure using the device.

The efficiency of the transformation using the electroporation devicesof the disclosure can result in at least 10% of the cells beingsufficiently porated to allow delivery of the biological molecule.Preferably, the efficiency of the transformation using theelectroporation devices of the disclosure can result in at least 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 75%, 80%, 85%, 90%, 95% orgreater of the cells being sufficiently porated to allow delivery of thebiological molecule.

Use of the Compound Electroporator in Exemplary Automated Multi-ModuleCell Processing Systems

As described above, the electroporation devices and modules are used ascomponents in an automated multi-module processing system. A generalexemplary embodiment of a multi-module cell processing system is shownin FIG. 7A. In some embodiments, the cell processing system 700 mayinclude a housing 760, a receptacle for introducing cells to betransformed or transfected 702, and a growth module 704. The cells to betransformed are transferred from a reservoir or receptacle to the growthmodule to be cultured until the cells hit a target OD. Once the cellshit the target OD, the growth module may cool or freeze the cells forlater processing or the cells may be transferred to a filtration module720 where the cells are rendered electrocompetent and concentrated. Thefiltration module 720 comprises e.g., a filter to treat the cells tomake them electrocompetent and concentrate the electrocompetent cells.In one example, 20 ml of cells+growth media is concentrated to 400 μlcells in 10% glycerol. Once the electrocompetent cells have beenconcentrated, the cells are transferred to an electroporation device tobe transformed with a desired nucleic acid. In addition to thereceptacle for receiving cells, the multi-module cell processing systemincludes a receptacle for storing the nucleic acids to be electroporatedinto the cells 706. The nucleic acids are transferred to theelectroporation module 708 (comprising, e.g., the electroporationdevice, liquid handling device, and electroporation station) whichalready contains the concentrated electrocompetent cells grown to thespecified OD, where the nucleic acids are introduced into the cells.Following electroporation, the transformed cells are transferred into,e.g., a recovery module 710. Here, the cells are allowed to recover fromthe electroporation procedure.

In some embodiments, after recovery the cells are transferred to astorage module 712 to be stored at, e.g., 4° C. or frozen. The cells canthen be retrieved from a retrieval module 714 and used for proteinexpression or further studies off-line. The automated multi-module cellprocessing system is controlled by a processor 750 configured to operatethe instrument based on user input or one or more scripts. The processor750 may control the timing, duration, temperature, and other operations(including, e.g., dispensing reagents) of the various modules of thesystem 700 as specified by one or more scripts. In addition to or as analternative to the one or more scripts, the processor may be programmedwith standard protocol parameters from which a user may select;alternatively, a user may select one or all parameters manually. Thescript may specify, e.g., the wavelength at which OD is read in the cellgrowth module, the target OD to which the cells are grown, the targettime at which the cells will reach the target OD, and the time, voltage,and/or wave form for electroporation. The processor may notify the user(e.g., via an application to a smart phone or other device) that thecells have reached the target OD as well as update the user as to theprogress of the cells in the cell growth module, electroporation device,filtration module, recovery module, etc. in the automated multi-modulecell processing system.

A second embodiment of an automated multi-module cell processing systemis shown in FIG. 7B. As with the embodiment shown in FIG. 7A, the cellprocessing system 770 may include a housing 760, a reservoir of cellsin, e.g., the reagent cartridge to be transformed or transfected 702,and a growth module (a cell growth device) 704. The cells to betransformed are transferred from a reservoir to the growth module to becultured until the cells hit a target OD. Once the cells hit the targetOD, the growth module may cool or freeze the cells for later processing,or the cells may be transferred to a filtration module 720 where thecells are rendered electrocompetent and concentrated to a volume optimalfor cell transformation/electroporation as described above in relationto FIG. 7A. Once concentrated, the cells are then transferred to theelectroporation module 708.

In addition to the reservoir for storing the cells, the reagentcartridge may include a reservoir for storing editing oligonucleotides716 and a reservoir for storing an expression vector backbone 718. Boththe editing oligonucleotides and the expression vector backbone aretransferred from the reagent cartridge to a nucleic acid assembly module720 (such as the nucleic acid assembly module described above), wherethe editing oligonucleotides are inserted into the expression vectorbackbone. The assembled nucleic acids may be transferred into anoptional purification module 722 for desalting and/or other purificationprocedures needed to prepare the assembled nucleic acids fortransformation. Once the processes carried out by the purificationmodule 722 are complete, the assembled nucleic acids are transferred tothe electroporation module 708, which already contains the cell culturegrown to a target OD. In electroporation module 708 the nucleic acidsare introduced into the cells. Following electroporation, the cells aretransferred into a combined recovery and editing module 712. Asdescribed above, in some embodiments the automated multi-module cellprocessing system 700 is a system that performs gene editing such as anRNA-direct nuclease editing system. For example, see in U.S. Ser. No.16/024,816, filed Jun. 30, 2018; U.S. Ser. No. 16/024,831, filed Jun.30, 2018, U.S. Ser. No. 62/657,651, filed Apr. 13, 2018; and U.S. Ser.No. 62/657,654, filed May 14, 2018. In the recovery and editing module710, the cells are allowed to recover post-transformation, and the cellsexpress the editing oligonucleotides that edit desired genes in thecells as described below.

Following editing, the cells are transferred to a storage module 714,where the cells can be stored at, e.g., 4° C. until the cells areretrieved for further study. The multi-module cell processing system iscontrolled by a processor 750 configured to operate the instrument basedon user input, as directed by one or more scripts, or as a combinationof user input or a script. The processor 750 may control the timing,duration, temperature, and operations of the various modules of thesystem 770 and the dispensing of reagents, and the time, voltage andwaveform for electroporation. The processor may be programmed withstandard protocol parameters from which a user may select, a user mayspecify one or more parameters manually or one or more scriptsassociated with the reagent cartridge may specify one or more operationsand/or reaction parameters. In addition, the processor may notify theuser (e.g., via an application to a smart phone or other device) thatthe cells have reached the target OD as well as update the user as tothe progress of the cells in the various modules in the automatedmulti-module system.

Certain embodiments of the multi-module processing system such as thesystem depicted in FIG. 7B include a nucleic acid assembly module (forexample, a Gibson Assembly® module, a Gap Repair module as used inyeast, or a module that performs, the polymerase chain reaction,ligation chain reaction, ligase detection reaction, ligation, circularpolymerase extension cloning, or other cloning methods) 730. The nucleicacid assembly module 730 is configured to assemble the nucleic acidsnecessary to facilitate genome editing events. In a nuclease-directedgenome editing system, a vector comprises one or more regulatoryelements operably linked to a polynucleotide sequence encoding a nucleicacid-guided nuclease. Thus, the nucleic acid assembly module 730 inthese embodiments is configured to assemble the expression vectorexpressing a nucleic acid guided nuclease. The nucleic acid assemblymodule 730 may be temperature controlled depending upon the type ofnucleic acid assembly used in the instrument. For example, when a GibsonAssembly® protocol is utilized, the module is configured to have theability to reach and hold 50° C. If PCR is performed as part of theautomated multi-module cell processing system, the nucleic acid assemblymodule is configured to thermocycle between temperatures. Thetemperatures and duration for maintaining temperatures can be effectedby a preprogrammed set of parameters (as dictated by a script orprogrammed into the processor), or manually controlled by the user usingthe processor.

As described above, in one embodiment the automated multi-module cellprocessing system 770 is a nuclease-directed genome editing system.Multiple nuclease-based systems exist for providing edits into a cell,and each can be used in either single editing systems as could beperformed in the automated system 700 of FIG. 7A; sequential editingsystems as could be performed in the automated system 780 of FIG. 7Cdescribed below, e.g., using different nuclease-directed systemssequentially to provide two or more genome edits in a cell; and/orrecursive editing systems as could be performed in the automated system780 of FIG. 7C, e.g. utilizing a single nuclease-directed system tointroduce two or more genome edits in a cell. Automatednuclease-directed processing systems use the nucleases to cleave thecell's genome, to introduce one or more edits into a target region ofthe cell's genome, or both. Nuclease-directed genome editing mechanismsinclude zinc-finger editing mechanisms (see Urnov et al., Nature ReviewsGenetics, 11:636-64 (2010)), meganuclease editing mechanisms (see Epinatet al., Nucleic Acids Research, 31(11):2952-62 (2003); and Arnould etal., Journal of Molecular Biology, 371(1):49-65 (2007)), and RNA-guidedediting mechanisms (see Jinek et al., Science, 337:816-21 (2012); andMali et al, Science, 339:823-26 (2013)). In particular embodiments, thenuclease editing system is an inducible system that allows control ofthe timing of the editing (see Campbell, Biochem J., 473(17): 2573-2589(2016); and Dow et al., Nature Biotechnology, 33390-94 (2015)). That is,when the cell or population of cells comprising a nucleic acid-guidednuclease encoding DNA is in the presence of the inducer molecule,expression of the nuclease can occur. The ability to modulate nucleaseactivity can reduce off-target cleavage and facilitate precise genomeengineering.

A third embodiment of a multi-module cell processing system is shown inFIG. 7C. This embodiment depicts an exemplary system 780 that performsrecursive gene editing on a cell population. As with the embodimentshown in FIGS. 7A and 7B, the cell processing system 780 may include ahousing 760, a reservoir in a reagent cartridge for storing cells to betransformed or transfected 702, and a cell growth module (a cell growthdevice) 704. The cells to be transformed are transferred from areservoir to the cell growth module to be cultured until the cells hit atarget OD. Once the cells hit the target OD, the growth module may coolor freeze the cells for later processing, or the cells may betransferred to a filtration module 730 where the cells are renderedelectrocompetent, and the volume of the cells may be reducedsubstantially. Once the cells have been concentrated to an appropriatevolume, the cells are transferred to electroporation module 708. Inaddition to the reservoir for storing cells, the multi-module cellprocessing system includes a reservoir for storing the vector comprisingediting oligonucleotides 706. The assembled nucleic acids aretransferred to the electroporation module 708, which already containsthe cell culture grown to a target OD. In the electroporation module708, the nucleic acids are electroporated into the cells. Followingelectroporation, the cells are transferred into a recovery module 724.In the recovery module 724, the cells are allowed to recoverpost-transformation.

The cells are transferred to a storage module 712, where the cells canbe stored at, e.g., 4° C. until the cells are retrieved for furtherstudy, or the cells are transferred to a second, optional, growth module726. Once the cells hit a target OD, the second growth module may coolor freeze the cells for later processing, or transfer the cells to,e.g., an editing module 728 where an inducible nuclease is expressed inthe cells, e.g., by introduction of heat or the introduction of aninducer molecule for expression of the nuclease. After editing, thecells are transferred to a separation and filtration module 730 wherethe cells are separated and/or concentrated from the editing solution inpreparation for transfer to electroporation module 708.

In electroporation module 708, the cells are transformed by a second setof editing oligos (or other type of oligos) and the cycle is repeateduntil the cells have been transformed and edited by a desired number of,e.g., editing oligonucleotides. As discussed above in relation to FIGS.7A and 7B, the multi-module cell processing system is controlled by aprocessor 750 configured to operate the instrument based on user inputor is controlled by one or more scripts including at least one scriptassociated with the reagent cartridge. The processor 750 may control thetiming, duration, and temperature of various processes; the dispensingof reagents; the time, voltage and waveform for electroporation; andother operations of the various modules of the system 780. For example,a script or the processor may control the dispensing of cells, reagents,vectors, and editing oligonucleotides; which editing oligonucleotidesare used for cell editing and in what order; the time, temperature andother conditions used in the recovery and expression module, thewavelength at which OD is read in the cell growth module, the target ODto which the cells are grown, and the target time at which the cellswill reach the target OD. In addition, the processor may be programmedto notify a user (e.g., via an application) as to the progress of thecells in the automated multi-module cell processing system.

While this invention is satisfied by embodiments in many differentforms, as described in detail in connection with preferred embodimentsof the invention, it is understood that the present disclosure is to beconsidered as exemplary of the principles of the invention and is notintended to limit the invention to the specific embodiments illustratedand described herein. Numerous variations may be made by persons skilledin the art without departure from the spirit of the invention. The scopeof the invention will be measured by the appended claims and theirequivalents. The abstract and the title are not to be construed aslimiting the scope of the present invention, as their purpose is toenable the appropriate authorities, as well as the general public, toquickly determine the general nature of the invention. In the claimsthat follow, unless the term “means” is used, none of the features orelements recited therein should be construed as means-plus-functionlimitations pursuant to 35 U.S.C. § 112, ¶ 6.

We claim:
 1. An automated multi-module cell processing systemcomprising: an electroporation module comprising an automated liquidhandling device and a multi-unit electroporation module, wherein eachelectroporation module comprises a housing configured to engage with apipette from the automated liquid handling device; an electroporationcuvette comprising an electroporation chamber defined by walls andwherein the electroporation cuvette is configured to receive a liquidfrom the automated liquid handling device; and a sipper in fluidcommunication with the electroporation chamber, wherein the sipper isconfigured for intake and output of a sample comprising cells to beelectroporated in the electroporation cuvette; and a growth module; arecovery module; and a processor.
 2. The automated multi-module cellprocessing system of claim 1, wherein the pipette is an automatic airdisplacement pipette.
 3. The automated multi-module cell processingsystem of claim 1, wherein the pipette is a multi-channel pipette. 4.The automated multi-module cell processing system of claim 1, whereinthe sample comprises bacterial cells.
 5. The automated multi-module cellprocessing system of claim 1, wherein the sample comprises mammaliancells.
 6. The automated multi-module cell processing system of claim 1,wherein the liquid comprises one or more materials to be electroporatedinto the cells selected from nucleic acids, peptides, proteins,hormones, cytokines, chemokines, drugs, or drug precursors.
 7. Theautomated multi-module cell processing system of claim 6, wherein theliquid comprises nucleic acids.
 8. The automated multi-module cellprocessing system of claim 1, wherein the liquid comprises at least onereagent of an RNA-directed nuclease editing system.
 9. The automatedmulti-module cell processing system of claim 1, wherein the processor isconfigured to read an optical density (OD) of the cells in the growthmodule.
 10. The automated multi-module cell processing system of claim9, wherein the processor is configured to instruct the growth modulethat the cells have reached the target OD.
 11. The automatedmulti-module cell processing system of claim 1, wherein the processor isconfigured to provide input for the pipette to move the cells into therecovery module after electroporation.
 12. The automated multi-modulecell processing system of claim 1, wherein the processor is configuredto provide input for each electroporation module for dispensing ofreagents via the automated liquid handling device.
 13. The automatedmulti-module cell processing system of claim 1, wherein the processor isconfigured to provide input for each electroporation module for thetime, voltage or waveform for electroporation.
 14. The automatedmulti-module cell processing system of claim 1, further comprising astorage module.
 15. The automated multi-module cell processing system ofclaim 14, wherein the storage module stores the cells at 4° C. or lowertemperatures.
 16. A method for electroporating a cell sample using theautomated multi-module cell processing system of claim 1, comprising thesteps of; moving the electroporation cuvette to a position to receivethe liquid from the automated liquid handling device; electroporatingthe sample comprising cells in the electroporation chamber of theelectroporation cuvette; moving the electroporation device to a positionto dispense the electroporated cell sample into the recovery module; anddispensing the cell sample from the electroporation chambers through thesippers and into the recovery module.
 17. The method claim 16, whereinthe recovery module comprises recovery medium to allow the pores of thecells to close and the cell membrane to recover from the electroporationprocess.
 18. A method for electroporating a cell sample using theautomated multi-module cell processing system of claim 1, comprising thesteps of; dispensing a sample from the growth module into theelectroporation cuvette through the sippers; moving the electroporationcuvette to a position to receive the liquid from the automated liquidhandling device; electroporating the sample comprising cells in theelectroporation chamber of the electroporation cuvette; moving theelectroporation device to a position to dispense the electroporated cellsample into the recovery module; and dispensing the cell sample from theelectroporation chambers through the sippers and into the recoverymodule.